High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy. More than 80% of HGSOC patients recur after frontline standard therapy, and complete and durable responses are rare. Therapies that improve compete tumor regression and prevent relapse are needed. Our GOALS are to i) identify which ovarian cancer genetic subsets will benefit from a new more effective genetically targeted therapy using primary tumor cultures, patient-derived xenograft models and human tumor samples from our clinical trial, ii) identify mechanisms of acquired resistance with genetic validation. We have identified a novel and highly active genotoxic therapy by co-inhibiting poly-ADP ribose polymerase (PARP) and ATR checkpoint kinase. Combination PARP inhibition with ATR inhibition (PARPi- ATRi) synergizes to specifically target and kill HGSOCs harboring common HGSOC-associated alterations, e.g. homologous recombination (HR) deficiency and Cyclin E overexpression. Our preliminary studies show that PARPi-ATRi in combination is especially effective in killing HGSOC cells with these alterations and causes regression of HR-deficient and Cyclin E overexpressing HGSOC patient-derived xenografts (PDXs).These results have stimulated a clinical trial of PARPi-ATRi combination in recurrent HGSOC (unselected patient population) to be led by the PI of this proposal. This will be the first trial evaluating PAPRi-ATRi combination in ovarian cancer. Here, we propose to validate in vitro findings and expand the use of our PDX model, a reliable HGSOC surrogate, to test the effectiveness of this novel PARPi-ATRi combination in killing HGSOCs that either overexpress Cyclin E-or have acquired PARPi-resistance, which together cause most deaths from HGSOC. Our clinical trial of PARPi-ATRi for treatment of recurrent HGSOC will be performed in parallel with the expanded preclinical mouse studies. This will allow us to correlate predictive biomarkers (HR-deficiency and Cyclin E overexpression) of response and potential resistance mechanisms (e.g., BRCA reversion mutations). This PARPi-ATRi combination therapy is promising; however, this treatment causes tumor regression initially in chemo-resistant Cyclin E-overexpressing tumors but acquired resistance ultimately develops. Using genomic and proteomics, we will integrate alterations discovered in the PAPRi-ATRi-resistant and sensitive PDXs with patient samples from the clinical trial to develop a candidate pool list. Candidate genes associated with resistance will be refined by prioritizing alterations found at both the gene and protein level and that are druggable. The top candidates will be validated in vitro using a targeted CRISPR knock-down approach. Defining markers of response and resistance will help stratify patient populations, optimize treatment strategies and identify new targets for future therapeutics based on HGSOC cancer genetics.